High Expression Levels Research Articles

Two lysin motif extracellular (LysMe) proteins are deployed in rice to facilitate arbuscular mycorrhizal symbiosis.

During arbuscular mycorrhizal (AM) symbiosis, plant innate immunity is modulated to a prime state to allow for fungal colonization. The underlying mechanisms remain to be further explored. In this study, two rice genes encoding LysM extracellular (LysMe) proteins were investigated. By obtaining OsLysMepro:GUS transgenic plants and generating oslysme1, oslysme2 and oslysme1oslysme2 mutants via CRISPR/Cas9 technique, OsLysMe genes were revealed to be specifically induced in the arbusculated cells and mutations in either gene caused significantly reduced root colonization rate by AM fungus Rhizophagus irregularis. Overexpression of OsLysMe1 or OsLysMe2 dramatically increased the colonization rates in rice and Medicago truncatula. The electrophoretic mobility shift assay and dual-luciferase reporter assay supported that OsLysMe genes are regulated by OsWRI5a. Either OsLysMe1 or OsLysMe2 can efficiently rescue the impaired AM phenotype of the mtlysme2 mutant, supporting a conserved function of LysMe across monocotyledonous and dicotyledonous plants. The co-localization of OsLysMe proteins with the apoplast marker SP-OsRAmy3A implies their probable localization to the periarbuscular space (PAS) during symbiosis. Relative to the fungal biomass marker RiTEF, some defense-related genes showed disproportionately high expression levels in the oslysme mutants. These data support that rice plants deploy two OsLysMe proteins to facilitate AM symbiosis, likely by diminishing plant defense responses.

The RNA binding protein IGF2BP2/IMP2 alters the cargo of cancer cell-derived extracellular vesicles supporting tumor-associated macrophages

BackgroundTumor cells release extracellular vesicles (EVs) that contribute to the polarization of macrophages towards tumor-associated macrophages (TAMs). High expression levels of the RNA binding protein IGF2BP2/IMP2 are correlated with increased tumor cell proliferation, invasion, and poor prognosis in the clinic. However, there is a lack of understanding of whether IMP2 affects the cargo of cancer cell-derived EVs, thereby modulating macrophage polarization.MethodsEVs were isolated from IMP2-expressing HCT116 parental cells (WT) and CRISPR/Cas9 IMP2 knockout (KO) cells. EVs were characterized according to MISEV guidelines, microRNA cargo was assessed by microRNA-Seq, and the protein cargo was analyzed by proteomics. Primary human monocyte-derived macrophages (HMDMs) were polarized by EVs, and the expression of genes and surface markers was assessed using qPCR and flow cytometry, respectively. Morphological changes of macrophages, as well as the migratory potential of cancer cells, were assessed by the Incucyte® system and macrophage matrix degradation potential by zymography. Changes in the metabolic activity of macrophages were quantified using a Seahorse® analyzer. For in vivo studies, EVs were injected into the yolk sac of zebrafish larvae, and macrophages were isolated by fluorescence-activated cell sorting.ResultsEVs from WT and KO cells had a similar size and concentration and were positive for 25 vesicle markers. The expression of tumor-promoting genes was higher in macrophages polarized with WT EVs than KO EVs, while the expression of TNF and IL6 was reduced. A similar pattern was observed in macrophages from zebrafish larvae treated in vivo. WT EV-polarized macrophages showed a higher abundance of TAM-like surface markers, higher matrix degrading activity, as well as a higher promotion of cancer cell migration. MicroRNA-Seq revealed a significant difference in the microRNA composition of WT and KO EVs, particularly a high abundance of miR-181a-5p in WT EVs, which was absent in KO EVs. Inhibitors of macropinocytosis and phagocytosis antagonized the delivery of miR-181a-5p into macrophages and the downregulation of the miR-181a-5p target DUSP6. Proteomics data showed differences in protein cargo in KO vs. WT EVs, with the differentially abundant proteins mainly involved in metabolic pathways. WT EV-treated macrophages exhibited a higher basal oxygen consumption rate and a lower extracellular acidification rate than KO EV-treated cells.ConclusionOur results show that IMP2 determines the cargo of EVs released by cancer cells, thereby modulating the EVs' actions on macrophages. Expression of IMP2 is linked to the secretion of EVs that polarize macrophages towards a tumor-promoting phenotype.

Metabolomic and transcriptomic analyses of monoterpene biosynthesis in Muscat and Neutral grape hybrids

Monoterpenes are typical aroma components of muscat grape (Vitis vinifera L.) cultivars. However, differences in the molecular mechanism of monoterpene biosynthesis between Muscat and non-Muscat grapes remain unclear. Here, comprehensive transcriptomic and metabolomic analyses of two F1 hybrids with different aroma profiles obtained from crosses between 'Muscat Hamburg' and 'Red Globe' grapes were performed to examine the unique monoterpene accumulation pattern and related candidate genes. The concentrations of most monoterpenes differed at various times, in the hybrid progeny, increasing from the pre-veraison stage to the veraison stage, followed by a decreasing trend from veraison to maturity. Within the F1 generation, the concentrations of accumulated monoterpenes were significantly higher in Muscat grapes than in neutral grapes. Furthermore, linalool, α-terpineol, and l-α-terpineol may be important odorants affecting the floral characteristics of Muscat grapes due to their high odor activity values. Transcriptional analysis identified three VvDXS (LOC100249323, LOC100268079, and LOC100247834), 1 VvHDS (LOC100257071), one VvMVK (LOC100251148), one VvHMGR (LOC100265082), and one VvGGPPS (LOC100257234) genes encoding proteins involved in the MEP and MVA pathways. Eleven TPS genes potentially involved in monoterpene regulation were also identified. Among them, VvTPS (LOC100853562) showed consistency with the accumulation of monoterpenes and had a high expression level; MYB (LOC100255488), GATA (LOC100262474), and ERF (LOC100255943, LOC100260401) were selected as candidate genes encoding transcription factors. The findings in this study provide new insights for further functional validation and investigation of genes related to monoterpene synthesis in Muscat aroma grapes.

Predicting CD27 expression and clinical prognosis in serous ovarian cancer using CT-based radiomics

BackgroundThis study aimed to develop and evaluate radiomics models to predict CD27 expression and clinical prognosis before surgery in patients with serous ovarian cancer (SOC).MethodsWe used transcriptome sequencing data and contrast-enhanced computed tomography images of patients with SOC from The Cancer Genome Atlas (n = 339) and The Cancer Imaging Archive (n = 57) and evaluated the clinical significance and prognostic value of CD27 expression. Radiomics features were selected to create a recursive feature elimination-logistic regression (RFE-LR) model and a least absolute shrinkage and selection operator logistic regression (LASSO-LR) model for CD27 expression prediction.ResultsCD27 expression was upregulated in tumor samples, and a high expression level was determined to be an independent protective factor for survival. A set of three and six radiomics features were extracted to develop RFE-LR and LASSO-LR radiomics models, respectively. Both models demonstrated good calibration and clinical benefits, as determined by the receiver operating characteristic (ROC) curves, calibration curves, and decision curve analysis. The LASSO-LR model performed better than the RFE-LR model, owing to the area under the curve (AUC) values of the ROC curves (0.829 vs. 0.736). Furthermore, the AUC value of the radiomics score that predicted the overall survival of patients with SOC diagnosed after 60 months was 0.788 using the LASSO-LR model.ConclusionThe radiomics models we developed are promising noninvasive tools for predicting CD27 expression status and SOC prognosis. The LASSO-LR model is highly recommended for evaluating the preoperative risk stratification for SOCs in clinical applications.

Genome-wide analysis of AP2/ERF transcription factors that regulate fruit development of Chinese prickly ash

BackgroundAP2/ERF is a large family of plant transcription factor proteins that play essential roles in signal transduction, plant growth and development, and responses to various stresses. The AP2/ERF family has been identified and verified by functional analysis in various plants, but so far there has been no comprehensive study of these factors in Chinese prickly ash. Phylogenetic, motif, and functional analyses combined with transcriptome analysis of Chinese prickly ash fruits at different developmental stages (30, 60, and 90 days after anthesis) were conducted in this study.ResultsThe analysis identified 146 ZbAP2/ERF genes that could be classified into 15 subgroups. The motif analysis revealed the presence of different motifs or elements in each group that may explain the functional differences between the groups. ZbERF13.2, ZbRAP2-12, and ZbERF2.1 showed high levels of expression in the early stages of fruit development. ZbRAP2-4, and ZbERF3.1 were significantly expressed at the fruit coloring stage (R2 and G2). ZbERF16 were significantly expressed at fruit ripening and expression level increased as the fruit continued to develop. Relative gene expression levels of 6 representative ZbAP2/ERFs assessed by RT-qPCR agreed with transcriptome analysis results.ConclusionsThese genes identified by screening can be used as candidate genes that affect fruit development. The results of the analysis can help guide future genetic improvement of Chinese prickly ash and enrich our understanding of AP2/ERF transcription factors and their regulatory functions in plants.

Follicular CD8+ T cells promote immunoglobulin production and demyelination in multiple sclerosis and a murine model

Emerging evidence underscores the importance of CD8+ T cells in the pathogenesis of multiple sclerosis (MS), but the precise mechanisms remain ambiguous. This study intends to elucidate the involvement of a novel subset of follicular CD8+ T cells (CD8+CXCR5+ T) in MS and an experimental autoimmune encephalomyelitis (EAE) murine model. The expansion of CD8+CXCR5+ T cells was observed in both MS patients and EAE mice during the acute phase. In relapsing MS patients, higher frequencies of circulating CD8+CXCR5+ T cells were positively correlated with new gadolinium-enhancement lesions in the central nervous system (CNS). In EAE mice, frequencies of CD8+CXCR5+ T cells were also positively correlated with clinical scores. These cells were found to infiltrate into ectopic lymphoid-like structures in the spinal cords during the peak of the disease. Furthermore, CD8+CXCR5+ T cells, exhibiting high expression levels of ICOS, CD40L, IL-21, and IL-6, were shown to facilitate B cell activation and differentiation through a synergistic interaction between CD40L and IL-21. Transferring CD8+CXCR5+ T cells into naïve mice confirmed their ability to enhance the production of anti-MOG35–55 antibodies and contribute to the disease progression. Consequently, CD8+CXCR5+ T cells may play a role in CNS demyelination through heightening humoral immune responses.

Prognostic prediction and diagnostic role of Rspondin 1 expression in esophageal squamous cell carcinoma.

Rspondin 1 (Rspo1), a protein family member featuring secreted furin-like domains, plays a pivotal role in cancer development and exhibits a positive correlation with tumor progression. However, its expression in esophageal squamous cell carcinoma (ESCC) is still unknown. Here, we assessed the correlation between Rspo1 and clinicopathological features of ESCC patients, and further investigated the potential role of Rspo1 in ESCC development and clinical outcomes. This was a pilot study. A total of 112 paraffin-embedded tumor samples from patients with ESCC, including 68 matched adjacent normal tissues, were collected post-surgery. Subsequently, tissue microarray (TMA) and immunohistochemistry (IHC) techniques were employed to assess the protein levels of Rspo1. All statistical analyses were performed with SPSS 20.0 (SPSS, Inc., Chicago, IL). We found that Rspo1 expression was significantly higher in ESCC than in adjacent normal tissues (P < 0.0001). Moreover, Rspo1 was highly expressed in ESCC tumor specimens and showed a significant correlation with the T classification of ESCC (P < 0.05). Additionally, our findings indicate a positive relationship between Rspo1 and survival time in ESCC. Patients exhibiting moderate to high levels of Rspo1 expression demonstrated superior survival outcomes compared to those with low expression (P = 0.0002). Our investigation has demonstrated that Rspo1 is upregulated in ESCC and exhibits a positive correlation with disease progression. Furthermore, we have observed a significant association between Rspo1 overexpression and improved patient survival rates, indicating its potential as a prognostic marker and therapeutic target for ESCC treatment.

Integrin αVβ1-activated PYK2 promotes the progression of non-small-cell lung cancer via the STAT3-VGF axis

BackgroundNon-small-cell lung cancer (NSCLC) accounts for 80–85% of all lung cancer and is the leading cause of cancer-related deaths globally. Although various treatment strategies have been introduced, the 5-year survival rate of patients with NSCLC is only 20–30%. Thus, it remains necessary to study the pathogenesis of NSCLC and develop new therapeutic drugs. Notably, PYK2 has been implicated in the progression of many tumors, including NSCLC, but its detailed mechanism remains unclear. In this study, we aimed to elucidate the mechanisms through which PYK2 promotes NSCLC progression.MethodsThe mRNA and protein levels of various molecules were measured using qRT-PCR, western blot (WB), and immunohistochemistry (IHC), respectively. We established stable PYK2 knockdown and overexpression cell lines, and CCK-8, EdU, and clonogenic assays; wound healing, transwell migration, and Matrigel invasion assays; and flow cytometry were employed to assess the phenotypes of tumor cells. Protein interactions were evaluated with co-immunoprecipitation (co-IP), immunofluorescence (IF)-based colocalization, and nucleocytoplasmic separation assays. RNA sequencing was performed to explore the transcriptional regulation mediated by PYK2. Secreted VGF levels were examined using ELISA. Dual-luciferase reporter system was used to detect transcriptional regulation site. PF4618433 (PYK2 inhibitor) and Stattic (STAT3 inhibitor) were used for rescue experiments. A public database was mined to analyze the effect of these molecules on NSCLC prognosis. To investigate the role of PYK2 in vivo, mouse xenograft models of lung carcinoma were established and examined.ResultsThe protein level of PYK2 was higher in human NSCLC tumors than in the adjacent normal tissue, and higher PYK2 expression was associated with poorer prognosis. PYK2 knockdown inhibited the proliferation and motility of tumor cells and caused G1-S arrest and cyclinD1 downregulation in A549 and H460 cells. Meanwhile, PYK2 overexpression had the opposite effect in H1299 cells. The siRNA-induced inhibition of integrins alpha V and beta 1 led to the downregulation of p-PYK2(Tyr402). Activated PYK2 could bind to STAT3 and enhance its phosphorylation at Tyr705, regulating the nuclear accumulation of p-STAT3(Tyr705). This further promoted the expression of VGF, as confirmed by RNA sequencing in a PYK2-overexpressing H1299 cell line and validated by rescue experiments. Two sites in promoter region of VGF gene were confirmed as binding sites of STAT3 by Dual-luciferase assay. Data from the TGCA database showed that VGF was related to the poor prognosis of NSCLC. IHC revealed higher p-PYK2(Tyr402) and VGF expression in lung tumors than in adjacent normal tissues. Moreover, both proteins showed higher levels in advanced TNM stages than earlier ones. A positive linear correlation existed between the IHC score of p-PYK2(Tyr402) and VGF. Knockdown of VGF inhibited tumor progression and reversed the tumor promoting effect of PYK2 overexpression in NSCLC cells. Finally, the mouse model exhibited enhanced tumor growth when PYK2 was overexpressed, while the inhibitors PF4618433 and Stattic could attenuate this effect.ConclusionsThe Integrin αVβ1-PYK2-STAT3-VGF axis promotes NSCLC development, and the PYK2 inhibitor PF4618433 and STAT3 inhibitor Stattic can reverse the pro-tumorigenic effect of high PYK2 expression in mouse models. Our findings provide insights into NSCLC progression and could guide potential therapeutic strategies against NSCLC with high PYK2 expression levels.

CD151-enriched migrasomes mediate hepatocellular carcinoma invasion by conditioning cancer cells and promoting angiogenesis

BackgroundThe tetraspanin family plays a pivotal role in the genesis of migrasomes, and Tetraspanin CD151 is also implicated in neovascularization within tumorous contexts. Nevertheless, research pertaining to the involvement of CD151 in hepatocellular carcinoma (HCC) neovascularization and its association with migrasomes remains inadequate.MethodsTo investigate the correlation between CD151 and migrasome marker TSPAN4 in liver cancer, we conducted database analysis using clinical data from HCC patients. Expression levels of CD151 were assessed in HCC tissues and correlated with patient survival outcomes. In vitro experiments were performed using HCC cell lines to evaluate the impact of CD151 expression on migrasome formation and cellular invasiveness. Cell lines with altered CD151 expression levels were utilized to study migrasome generation and in vitro invasion capabilities. Additionally, migrasome function was explored through cellular aggregation assays and phagocytosis studies. Subsequent VEGF level analysis and tissue chip experiments further confirmed the role of CD151 in mediating migrasome involvement in angiogenesis and cellular signal transduction.ResultsOur study revealed a significant correlation between CD151 expression and migrasome marker TSPAN4 in liver cancer, based on database analysis of clinical samples. High expression levels of CD151 were closely associated with poor survival outcomes in HCC patients. Experimentally, decreased CD151 expression led to reduced migrasome generation and diminished in vitro invasion capabilities, resulting in attenuated in vivo metastatic potential. Migrasomes were demonstrated to facilitate cellular aggregation and phagocytosis, thereby promoting cellular invasiveness. Furthermore, VEGF-enriched migrasomes were implicated in signaling and angiogenesis, accelerating HCC progression.ConclusionsIn summary, our findings support the notion that elevated CD151 expression promotes migrasome formation, and migrasomes play a pivotal role in the invasiveness and angiogenesis of liver cancer cells, thereby facilitating HCC progression. This finding implies that migrasomes generated by elevated CD151 expression may constitute a promising high-priority target for anti-angiogenic therapy in HCC, offering crucial insights for the in-depth exploration of migrasome function and a renewed comprehension of the mechanism underlying liver cancer metastasis.

In silico approach to investigate the potential HKT gene responsive to salt stress in rice

Rice is frequently subjected to various environmental stresses, resulting in significant production losses, with drought and salinity are the leading causes of plant damage globally. This study aims to characterize and understand the function of rice high-affinity potassium transporters (HKTs) genes in response to salinity stress. Initially, the genome-wide analysis was undertaken to reveal the evolutionarily conserved function of the OsHKT in higher plants. To investigate the transcription level of OsHKT during the vegetative and reproductive stages, two microarray datasets (GSE19024 and GSE3053) were analyzed, and salt-treated samples were subsequently evaluated using real-time PCR. Differentially expressed genes (DEGs) were identified from microarray datasets (GSE41650 and GSE14403), followed by constructing a DEG network that highlighted interaction partners of the OsHKTs. Genome mining of rice revealed 9 HKT genes, namely OsHKT1;1–1;5 and OsHKT2;1–2;4. These genes exhibited a well-conserved domain structure called TrkH. Comprehensive phylogenetic and motif analyses clustered genes encoding HKT proteins into seven monophyletic groups, and the motifs were relatively conserved. Ka/Ks ratios indicated a high degree of purifying selection during evolutionary time. Gene ontology findings suggested the involvement of OsHKT in stress response. Besides, several CRE motifs in the promoter regions of OsHKT have demonstrated their potential roles in abiotic stress responses. Furthermore, we analyzed the top 250 significant DEGs from the two datasets (p-value < 0.05; fold two change ≥ 1 or ≤ − 1) to evaluate the relationship among the DEGs and HKTs. Three co-expressed OsHKT genes were discovered to be upregulated in seedlings under salinity treatment, including OsP5CS2, OsHAK1, and OsNHX2, whereas OsP5CS1 and OsHAK27 were downregulated. The transcripts of OsHKT were found to be differentially expressed in a tissue-specific manner. Analysis of microarray datasets validated by real-time PCR shows that OsHKT1;5 had a higher expression level, followed by OsHKT1;1, OsHKT1;3, and OsHKT2;1 after salinity treatment. In addition, several micro-RNA targets in rice HKT genes regulate their expression in response to stress. This study paves the way for future investigation on genes and miRNA-target interaction in plants under environmental stresses, offering potential strategies to enhance stress tolerance in crops via targeted ion transport modification.

Chemical Proteomic Profiling of Protein Dopaminylation in Colorectal Cancer Cells.

Histone dopaminylation is a newly identified epigenetic mark that plays a role in the regulation of gene transcription, where an isopeptide bond is formed between the fifth amino acid of H3 (i.e., glutamine) and dopamine. Recently, we developed a chemical probe to specifically label and enrich histone dopaminylation via bioorthogonal chemistry. Given this powerful tool, we found that histone H3 glutamine 5 dopaminylation (H3Q5dop) was highly enriched in colorectal tumors, which could be attributed to the high expression level of its regulator, transglutaminase 2 (TGM2), in colon cancer cells. Due to the enzyme promiscuity of TGM2, nonhistone proteins have also been identified as dopaminylation targets; however, the dopaminylated proteome in cancer cells still remains elusive. Here, we utilized our chemical probe to enrich dopaminylated proteins from colorectal cancer cells in a bioorthogonal manner and performed the chemical proteomics analysis. Therefore, 425 dopaminylated proteins were identified, many of which are involved in nucleic acid metabolism and transcription pathways. More importantly, a number of dopaminylation sites were identified and attributed to the successful application of our chemical probe. Overall, these findings shed light on the significant association between cellular protein dopaminylation and cancer development, further suggesting that targeting these pathways may become a promising anticancer strategy.

Minimizing IP issues associated with gene constructs encoding the Bt toxin - a case study

BackgroundAs part of a publicly funded initiative to develop genetically engineered Brassicas (cabbage, cauliflower, and canola) expressing Bacillus thuringiensis Crystal (Cry)-encoded insecticidal (Bt) toxin for Indian and Australian farmers, we designed several constructs that drive high-level expression of modified Cry1B and Cry1C genes (referred to as Cry1BM and Cry1CM; with M indicating modified). The two main motivations for modifying the DNA sequences of these genes were to minimise any licensing cost associated with the commercial cultivation of transgenic crop plants expressing CryM genes, and to remove or alter sequences that might adversely affect their activity in plants.ResultsTo assess the insecticidal efficacy of the Cry1BM/Cry1CM genes, constructs were introduced into the model Brassica Arabidopsis thaliana in which Cry1BM/Cry1CM expression was directed from either single (S4/S7) or double (S4S4/S7S7) subterranean clover stunt virus (SCSV) promoters. The resulting transgenic plants displayed a high-level of Cry1BM/Cry1CM expression. Protein accumulation for Cry1CM ranged from 5.18 to 176.88 µg Cry1CM/g dry weight of leaves. Contrary to previous work on stunt promoters, we found no correlation between the use of either single or double stunt promoters and the expression levels of Cry1BM/Cry1CM genes, with a similar range of Cry1CM transcript abundance and protein content observed from both constructs. First instar Diamondback moth (Plutella xylostella) larvae fed on transgenic Arabidopsis leaves expressing the Cry1BM/Cry1CM genes showed 100% mortality, with a mean leaf damage score on a scale of zero to five of 0.125 for transgenic leaves and 4.2 for wild-type leaves.ConclusionsOur work indicates that the modified Cry1 genes are suitable for the development of insect resistant GM crops. Except for the PAT gene in the USA, our assessment of the intellectual property landscape of components presents within the constructs described here suggest that they can be used without the need for further licensing. This has the capacity to significantly reduce the cost of developing and using these Cry1M genes in GM crop plants in the future.

Novel TROP2-targeted CAR T cells with high specificity and sustained efficacy for solid tumors in animal models.

e14563 Background: CAR T cell therapy, successful in hematological malignancies, encounters challenges against solid tumors due to immune exhaustion and senescence within the tumor microenvironment. The imperative need for an optimal tumor target antigen is evident in advancing CAR T therapy. TROP2, a membrane-associated glycoprotein, is highly expressed in diverse cancers, including cervical, bladder, esophageal, ovarian, and endometrial cancers. TROP2 overexpression, approaching 100% in cervical and hormone-resistant prostate cancer, correlates inversely with prognosis and survival, presenting TROP2 as a promising CAR T therapy target. This study engineered the novel TROP2 antibody R4702, featuring a distinct binding epitope from Sacituzumab and Datopotamab, to develop TROP2-specific CAR T cells and evaluated their efficacy against a spectrum of solid tumors. Methods: Expansion of CAR T Cells and Cytotoxicity: CAR T cells were created through lentivirus-mediated genetic engineering with the TROP2 antibody, followed by 10 days of expansion. T cell differentiation was evaluated, and luciferase-encoding target cells incubated with varying CAR T cell ratios overnight were quantified luciferase activity. In Vivo Efficacy and Persistence: CAR T cells were adoptively transferred into ASID mice with tumors. In vivo tumor imaging used luciferase activity, and calipers monitored tumor size weekly. CAR T cell persistence assessed their ability to suppress rechallenged tumor growth. Results: R4702, a novel TROP2-specific antibody, displayed high binding affinity to a distinct epitope. Post- ex vivo expansion, R4702 CAR T cells showed a substantial increase (100 to 200-fold) over 10 days, with a high expression level (50~80%) of R4702 CAR on T cells. Over 60% of R4702 CAR T cells displayed a less differentiated status, characterized by a CD62L+ population comprising TSCM and TCM. In vitro cytotoxicity revealed potent and specific efficacy against various TROP2-positive cancer cell lines, with no observed killing activity against TROP2-negative cells. In vivo efficacy of R4702 CAR T cells demonstrated 100% tumor growth inhibition within 30 days post-treatment across diverse xenograft models. Importantly, R4702 CAR T cells exhibited enduring anti-tumor efficacy against successive tumor challenges in xenograft models of pancreatic, gastric, NSCLC, TNBC, and ovarian cancers. In a head-to-head comparison with Sacituzumab and Datopotamab CAR T, R4702 demonstrated superior anti-tumor efficacy to Sacituzumab and comparable efficacy with Datopotamab in the BxPC-3 CDX model of pancreatic cancer. Conclusions: A novel TROP2-specific R4702 CAR T cell, developed, showcased robust tumor-killing capabilities. The anti-tumor activity persisted upon repeated tumor challenges across various xenograft models, underscoring the potential of R4702 CAR T cells as a promising therapeutic strategy.

Odorant-binding protein CrufOBP1 in Cotesia ruficrus females plays a pivotal role in the detection of Spodoptera frugiperda larvae

Odorant-binding protein CrufOBP1 in Cotesia ruficrus females plays a pivotal role in the detection of Spodoptera frugiperda larvae

Nectin-4 expression in primary breast cancer and associated clinical outcomes.

570 Background: Antibody-drug conjugates (ADC) selectively deliver chemotherapy to tumor cells expressing a target antigen. Currently approved ADCs in breast cancer target HER2 or Trop-2. Enfortumab vedotin, an ADC targeting Nectin-4, a cell adhesion molecule, is approved in advanced/metastatic urothelial cancer. This study evaluated the expression landscape of Nectin-4 in breast cancer and its association with clinical outcomes. Methods: Processed mRNAseq data was assessed for expression of the gene encoding Nectin-4 in primary breast cancer tissue and adjacent normal breast tissue from The Cancer Genome Atlas (TCGA), and normal tissue from the Genotype-Tissue Expression (GTEx) Project. Low gene expression was determined as &lt;100 transcripts per million (TPM). TCGA samples had been annotated for putative copy-number alterations using GISTIC 2.0; genes with a score of 2 were marked as amplified. Multivariable Cox regression analysis was used to analyze the association of low vs medium/high gene expression with disease-free survival (DFS) and overall survival (OS), adjusting for age, stage, and subtype. Results: RNA-Seq data was available for 1086 patients with primary breast cancer, including known hormone receptor positive (HR+)/HER2- (n=530), HER2+ (n=112), and triple negative breast cancer (TNBC) (n=120). NECTIN4was amplified in 15% of cases (n=165): 15% (n=78) in HR+, 15% (n=17) in HER2+ and 21% (n=25) in TNBC. NECTIN4 expression was medium/high in 356 (33%) patients: 149 (28%) HR+/HER2- (median 77.8 TPM, IQR 50.1-116.5), 32 (29%) HER2+ (median 73.7 TPM, IQR 51.9-107.8), and 59 (49%) TNBC (median 99.7 TPM, IQR 57.8-148.3). Median expression in normal breast tissue was 47 TPM (TCGA) &amp; 16.74 TPM (GTEx), and highest in normal skin at 205 TPM. In multivariable analysis, medium/high NECTIN4 expression was associated with worse OS (HR 1.96, 95% CI 1.32-2.99, p&lt;0.001). As a continuous variable, increasing NECTIN4 expression levels were associated with worse OS. After correcting for age, stage and subtype, for each 10-unit increase in NECTIN4TPM, the risk of death increased by 5% (HR 1.0050, 95% CI 1.0028 - 1.0072, p &lt; 0.001). Conclusions: NECTIN4 expression was higher in TNBC and associated with worse overall survival. The high expression level in normal skin possibly contributes to the cutaneous toxicity associated with enfortumab vedotin approval and requires careful consideration as ADCs targeting Nectin-4 are developed to target breast cancer.

TLR21 is involved in the NF-κB and IFN-β pathways in largemouth bass (Micropterus salmoides) and interacts with TRIF but not with the Myd88 adaptor

Toll-like receptors (TLRs) are pattern recognition receptors that trigger host immune responses against various pathogens by detecting evolutionarily conserved pathogen-associated molecular patterns (PAMPs). TLR21 is a member of the Toll-like receptor family, and emerging data suggest that it recognizes unmethylated CpG DNA and is considered a functional homologue of mammalian TLR9. However, little is known regarding the role of TLR21 in the fish immune response. In the present study, we isolated the cDNA sequence of TLR21 from the largemouth bass (Micropterus salmoides) and termed it MsTLR21. The MsTLR21 gene contained an open reading frame (ORF) of 2931 bp and encodes a polypeptide of 976 amino acids. The predicted MsTLR21 protein has two conserved domains, a conserved leucine-rich repeats (LRR) domain and a C-terminal Toll-interleukin (IL) receptor (TIR) domain, similar to those of other fish and mammals. In healthy largemouth bass, the TLR21 transcript was broadly expressed in all the examined tissues, with the highest expression levels in the gills. After challenge with Nocardia seriolae and polyinosinic polycytidylic acid (Poly[I:C]), the expression of TLR21 mRNA was upregulated or downregulated in all tissues tested. Overexpression of TLR21 in 293T cells showed that it has a positive regulatory effect on nuclear factor-kappaB (NF-κB) and interferons-β (IFN-β) activity. Subcellular localisation analysis showed that TLR21 was expressed in the cytoplasm. We performed pull-down assays and determined that TLR21 did not interact with myeloid differentiation primary response gene 88 (Myd88); however, it interacted with TIR domain-containing adaptor inducing interferon-β (TRIF). Taken together, these findings suggest that MsTLR21 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.

Molecular subtyping of skin cutaneous melanoma based on inflammatory response

The inflammatory response plays a crucial role in determining the prognosis and therapeutic response of skin cutaneous melanoma (SKCM). However, the molecular subtypes based on the inflammatory response and their clinical significance in SKCM have not been extensively studied. Clustering analyses to identify inflammation subtypes of SKCM based on the expression levels of inflammation response gene. We identified three subtypes: Inflammation_H, Inflammation_M, and Inflammation_L, which offer a more nuanced understanding of the complex relationship between inflammation and SKCM. The Inflammation_H subtype is associated with the most favourable prognosis, and is characterised by high levels of immune infiltrates and PD-L1 expression, low levels of stemness, high differentiation, and high genomic stability. In contrast, the Inflammation_L subtype has the least favourable prognosis, with the lowest levels of immune infiltrates and PD-L1 expression, high levels of stemness, low differentiation, and low genomic stability. In addition, the Inf-score, which is a linear risk scoring model based on the expression levels of inflammatory response genes, can be a useful tool for clinicians to assess SKCM prognosis and guide therapeutic decisions. This scoring model shows promise for clinical use in predicting patient outcomes and helps clinicians tailor treatments for individual patients.In conclusion, these findings represent a significant contribution to our understanding of the molecular subtypes of SKCM based on the levels of inflammatory response genes and their potential clinical significance. However, further studies are necessary to validate these findings and explore the underlying mechanisms of different subtypes.

Expression and Prognostic Significance of LAG-3, TIGIT, VISTA, and IDO1 in Endometrial Serous Carcinoma

Endometrial serous carcinoma (ESC) is an uncommon, aggressive type of endometrial cancer. While immune checkpoint blockade has emerged as a promising treatment option for endometrial carcinomas, research on the expression of immune checkpoints that could serve as prospective immunotherapy targets in ESC is limited. We examined the prevalence and prognostic value of LAG-3, TIGIT, VISTA, and IDO1 in 94 cases of ESC and correlated their expression with CD8+ and FOXP3+ tumor infiltrating lymphocytes (TIL). We observed a positive correlation between LAG-3, TIGIT and VISTA expressed on immune cells, and between these markers and CD8+ and FOXP3+ TIL density. In Kaplan-Meier survival analysis, tumors with high levels of LAG-3 and TIGIT expression had better progression free survival (PFS) and overall survival (OS) than those with lower levels of expression (LAG-3: PFS, p=0.03, OS, p=0.04; TIGIT: PFS, p=0.01, OS, p=0.009). In multivariate analysis, only high TIGIT expression was of independent prognostic value for better overall survival. VISTA expression in immune or tumor cells, and IDO1 expression in tumor cells, did not show a significant association with survival. Our data indicate that LAG-3, TIGIT, and VISTA immune checkpoints have roles in the microenvironment of ESC, and their expression patterns highlight the complex interactions among the different components of this system. High levels of these markers, together with high CD8+ TIL, suggests the potential immunogenicity of a subset of these tumors. Further studies are needed to elucidate the roles of various immune components in the ESC microenvironment and their association with intrinsic tumor properties.

Cas9 ribonucleoproteins and single-strand oligodeoxynucleotides enable targeted insertional mutagenesis in Ulva

Ulva is a green seaweed that is widely distributed in coastal areas around the world, and genomic information has been reported for several species. However, research on reverse genetics methods is still in its early stages. Although our group has recently achieved efficient knockout of Ulva by introducing Cas9 ribonucleoprotein (RNP) through the PEG method, a precise knock-in transformation method is needed to understand gene and protein functions. Here, we report the development of a targeted insertional mutagenesis method by transfection of Cas9 RNP, donor dsDNA, and single-stranded DNAs homologous to the DSB site created by Cas9 and donor dsDNA. The target gene was RbcS, a well-known high-expression gene, and we attempted to knock in the EGFP or 2A peptide-EGFP sequence at the C-terminus region. This method was also applied to the adenine phosphoribosyltransferase (APT) gene, enabling selection by the toxic compound 2-fluoroadenine. A longer DNA fragment (2.6 kb) expressing EGFP under the RbcS promoter was inserted at the DSB site on the APT locus. As a result, EGFP knock-in transformants were obtained, and the transformant progeny exhibited high EGFP expression levels for several generations. Our method greatly advances the genetic engineering and functional analysis of target genes in Ulva.

Azoxymethane-induced carcinogenesis-like model of mouse intestine and mouse embryonic stem cell-derived intestinal organoids.

Tumor modeling using organoids holds potential in studies of cancer development, enlightening both the intracellular and extracellular molecular mechanisms behind different cancer types, biobanking, and drug screening. Intestinal organoids can be generated in vitro using a unique type of adult stem cells which are found at the base of crypts and are characterized by their high Lgr5 expression levels. In this study, we successfully established intestinal cancer organoid models by using both the BALB/c derived and mouse embryonic stem cells (mESCs)-derived intestinal organoids. In both cases, carcinogenesis-like model was developed by using azoxymethane (AOM) treatment. Carcinogenesis-like model was verified by H&E staining, immunostaining, relative mRNA expression analysis, and LC/MS analysis. The morphologic analysis demonstrated that the number of generated organoids, the number of crypts, and the intensity of the organoids were significantly augmented in AOM-treated intestinal organoids compared to non-AOM-treated ones. Relative mRNA expression data revealed that there was a significant increase in both Wnt signaling pathway-related genes and pluripotency transcription factors in the AOM-induced intestinal organoids. We successfully developed simple carcinogenesis-like models using mESC-based and Lgr5 + stem cell-based intestinal organoids. Intestinal organoid based carcinogenesi models might be used for personalized cancer therapy in the future.